Nonalcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis ...

International Journal of

Molecular Sciences

Review

Nonalcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis: Current Issues and Future Perspectives in Preclinical and Clinical Research

Clarissa Berardo , Laura Giuseppina Di Pasqua * , Marta Cagna, Plinio Richelmi, Mariapia Vairetti and Andrea Ferrigno *

Unit of Cellular and Molecular Pharmacology and Toxicology, Department of Internal Medicine and Therapeutics, University of Pavia, 27100 Pavia, Italy; clarissa.berardo01@universitadipavia.it (C.B.); marta.cagna02@universitadipavia.it (M.C.); plinio.richelmi@unipv.it (P.R.); mariapia.vairetti@unipv.it (M.V.) * Correspondence: lauragiuseppin.dipasqua01@universitadipavia.it (L.G.D.P.); andrea.ferrigno@unipv.it (A.F.);

Tel.: +39-0382-986-451 (L.G.D.P.)

Received: 24 November 2020; Accepted: 16 December 2020; Published: 17 December 2020

Abstract: Nonalcoholic fatty liver disease (NAFLD) is a continuum of liver abnormalities often starting as simple steatosis and to potentially progress into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Because of its increasing prevalence, NAFLD is becoming a major public health concern, in parallel with a worldwide increase in the recurrence rate of diabetes and metabolic syndrome. It has been estimated that NASH cirrhosis may surpass viral hepatitis C and become the leading indication for liver transplantation in the next decades. The broadening of the knowledge about NASH pathogenesis and progression is of pivotal importance for the discovery of new targeted and more effective therapies; aim of this review is to offer a comprehensive and updated overview on NAFLD and NASH pathogenesis, the most recommended treatments, drugs under development and new drug targets. The most relevant in vitro and in vivo models of NAFLD and NASH will be also reviewed, as well as the main molecular pathways involved in NAFLD and NASH development.

Keywords: non-alcoholic fatty liver disease; metabolic syndrome; steatohepatitis; hepatocellular carcinoma; steatosis

1. Introduction

In the last decades, a rise in the diffusion of chronic liver pathologies has been observed; among them, one of the most insidious is nonalcoholic fatty liver disease (NAFLD) [1]. NAFLD is characterized by the intracellular deposition of lipids in hepatocytes, often associated with a wide spectrum of metabolic abnormalities, such as dyslipidemia, hypertension, insulin resistance and diabetes; these features are collectively known as the manifestation of metabolic syndrome [2]. Under the umbrella term of "NAFLD", adopted in 1986 [3], a wide range of pathological conditions are comprised: simple steatosis (fat accumulation in the hepatic parenchyma), non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. In a three-year period, 20?30% of patients with simple steatosis have been found to progress toward NASH, a more severe condition in which fat accumulation is accompanied by inflammation and oxidative stress [4]. The resulting chronic inflammatory state may develop into fibrosis, cirrhosis and hepatocellular carcinoma. Patients suffering from simple steatosis present higher life expectancy than those affected by NASH, who also incur in cardiovascular damage [2]. Recent studies have demonstrated that NASH is one of the most important causes of liver transplantation in the USA, and it will become the leading cause for the request of liver donors in the next decades [5].

Int. J. Mol. Sci. 2020, 21, 9646; doi:10.3390/ijms21249646

journal/ijms

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Recently, the name used to define NAFLD has been questioned. On one hand, the use of the term "non" has been judged to diminish the importance of the condition; on the other hand, "non-alcoholic fatty liver disease" fits the definition of "non-communicable diseases" estimated to cause >70% of global death [6]. In this context, a panel of experts recommended renaming NAFLD into metabolic (dysfunction)-associated fatty liver disease (MAFLD) [7], concomitantly with the adoption of new diagnostic criteria [8]. At the moment, a general consensus has not been achieved as some reason for criticism has been found in the proposed diagnostic criteria [9]; even though the old negative definition is generally considered outdated, over-emphasizing the metabolic dysfunctions in MAFLD might lead to the underestimation of the impact of steatosis itself in a significant group of patients at risk of disease progression [10,11].

In this review, we will discuss NAFLD pathogenesis, its progression to NASH, the currently recommended treatments, new drug candidates under development and potential new drug targets. The most relevant in vitro and in vivo models of NAFLD and NASH will also be reviewed, as well as the main molecular pathways involved in NAFLD and NASH development.

2. Methods

A search through PubMed was performed to identify relevant articles published until 18 November 2020. Search terms included steatosis and nonalcoholic steatohepatitis in combination with epidemiology, management, pathogenesis, fibrosis, cirrhosis, liver transplantation, hepatocellular carcinoma, experimental models, new drug targets. Additional relevant articles were identified from citations referenced in other articles, if they did not appear in the original search.

3. Epidemiology

It has been recently estimated that NAFLD affects 24% of the world's population. The highest prevalence rate has been registered in South America (31%), followed by Middle East (32%), Asia (27%), USA (24%) and Europe (23%). The lowest rate, instead, was observed in Africa (14%). Based on studies conducted from 2005 to 2015, the prevalence of NAFLD is increasing and a similar trend was observed also for NASH [1]. In agreement with its metabolic origin, 42% of NAFLD subjects show metabolic syndrome (MetS) [1,12]. Metabolic syndrome (MetS) is defined as a cluster of metabolic disorders including abdominal obesity, hypertension, dyslipidemia and impaired glycemia [13]. MetS is highly prevalent worldwide, as well as its related metabolic disorders [14]. The prevalence of NAFLD was found to be significantly greater (43.2%) in patients with MetS when compared with its prevalence in the general population. In addition, the prevalence of advanced hepatic fibrosis, which was found to be 6.6% in people with mild to severe steatosis, rose to 30.3% in people with five MetS abnormalities [15]. In obese people, NAFLD recurrence ranges from 60 to 95% [12]; in a cohort study in which subjects were observed over a period of approximately 4 years, visceral adipose tissue, and not subcutaneous adipose tissue, was associated with higher risk of NAFLD with an adjusted hazard ratio of 2.23 (95% CI 1.28?3.89) [16]. The association between type-2 diabetes (T2D) and NAFLD has recently been investigated in two meta-analyses. The first one involved 35,599 T2D patients from twenty-four studies, 20,264 of them were diagnosed with NAFLD; the pooled prevalence of NAFLD in T2D patients was 59.67% (95% CI 54.31?64.92), which rose to 77.87% (95% CI 65.51?88.14) in those with obesity [17]. In another meta-analysis, obtained combining data from 80 studies (49,419 individuals), a global NAFLD prevalence of 55.5% (95% CI 47.3?63.7) was found among patients with T2D; in Europe, a prevalence of 68% (95% CI 62.1?73.0) was found, followed by West Asia (67.29%; 95% CI 60.39?73.61%), South Asia (57.87%; 95% CI 52.87?62.68%), Latin America (56.83%; 95% CI 34.05?76.98%), East Asia (52.04%; 95% CI 45.37?58.55%), United States (51.77%; 95% CI 31.33?71.64%) and Africa (30.39%; 95% CI 11.64?67.09%) [18].

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In addition to the metabolic risk factors, sex and age also have a major influence on the risk of NAFLD [19]. With regard to the influence of sex, it has been observed that inconsistent findings were previously found; however, the majority of the studies did not clearly distinguish pre- and post-menopausal women, resulting in contradictory results. In more recent studies, men were found to be associated with a higher risk for advanced fibrosis in comparison with pre-menopausal women. Differently, both sexes display similar severity of fibrosis when women after menopause were considered, suggesting that estrogen may protect from advanced liver injury (reviewed by Lonardo et al., (2020)) [20]. In addition, in a study on obese individuals approximately 41 years old, NASH has been independently associated with the male sex [21]. In older patients NAFLD is more prevalent; in fact, the majority of patients were diagnosed between 40 to 50 years of age. Moreover, older age is associated with a higher risk of NASH and fibrosis [22]. However, it should be noted that, as a result of a constant increase in obesity rate, NAFLD is currently the most common cause of liver disease in children, ranging from steatosis, to NASH and fibrosis [23].

Studies based on liver biopsies have shown that patients with NASH are at the highest risk for cirrhosis. In a study, after 12 years of follow-up, risk of liver-related mortality was 6 times higher in patients with NASH than in non-NASH NAFLD patients [24]. Similar conclusions have been obtained in multiple other studies [25?27]. Long-term outcome of patients with NAFLD diagnosed by non-invasive techniques also provided evidences that T2D and MetS may accelerate disease progression and increase the risk of liver-related mortality [28?30]. Advanced fibrosis was found to induce a 2- or 3-fold increase in the risk for cardiovascular mortality in NAFLD patients, supposedly because of a proinflammatory environment and endothelial dysfunction observed in patients with NAFLD [31]. NAFLD and NASH are also well-known risk factors for developing HCC [19]. Patients with NAFLD-related fibrosis (stages 3 and 4) have approximately a 7 times higher risk for developing HCC; however, it is interesting to note that also NAFLD patients with no evidence of fibrosis may develop HCC [32]; in fact, NASH-related HCC is increasingly being seen in clinical setting, as indirectly demonstrated by the increasing number of patients being listed for liver transplantation related to HCC [19].

The variable course of NAFLD progression poses a clinical challenge that needs to be addressed by better characterizing the multiple modifiers of the disease, including the role of genetic polymorphisms, family and personal history, alcohol or drug consumption, diet, physical activity. Various topics have been proposed as relevant research priorities [33], such as the role of sex, depression and anxiety, endocrine disorders, chronic obstructive pulmonary disease and sleep apnoea syndrome in the pathogenesis of NAFLD, as well as the therapeutic role of intermittent fasting and anticoagulation [33].

4. Diagnosis

Considering the high prevalence of this pathology, its severe stages of progression and the difficulty encountered by the clinicians in identifying it, the development of reliable, non-invasive diagnostic techniques, able to recognize the disease in both its early and advanced stages, possibly avoiding liver biopsy, is very pressing [34].

NAFLD is often asymptomatic; in fact, patients usually discover to be affected by NAFLD only incidentally, during routine laboratory examination, when the hepatic panel reveals increased transaminase serum levels. In addition, about 80% of patients show normal ALT levels that also tend to decrease in the pathology progression from the stage of fibrosis to cirrhosis, making it unhelpful in the diagnosis and causing errors in the evaluation of clinicians, who may neglect patients potentially at risk [35].

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The joint European Association for the Study of the Liver, European Association for the Study of Diabetes and the European Association for the Study of Obesity (EASL-EASD-EASO) guidelines for the management of nonalcoholic fatty liver disease recommend that simple steatosis should be diagnosed by imaging, in particular ultrasonography (US), because it is widely used and less expensive than the gold standard, magnetic resonance imaging (MRI). US, MRI or computed tomography (CT) were considered low-performance techniques for the detection of mild steatosis; however, some semi-quantitative scores (i.e., Hamaguchi score, US FLI and hepatorenal steatosis index) have been proposed to improve US performance (reviewed by Ferraioli et al., 2019) [36]. These techniques are currently considered first-line diagnostic tests in case of moderate and severe steatosis, providing also information about the hepatobiliary system. Nevertheless, the serum biomarkers investigation is the method of choice when a large scale screening is needed because the costs of imaging techniques are not sustainable [37].

Since liver enzymes and imaging are not able to predict the onset and presence of NASH [34], novel serum markers have been taken into account, such as the intermediate filament protein cytokeratin 18 (CK18), a fragment obtained after caspase-3-mediated cleavage of different substrates during the apoptotic process, one of the main mechanisms induced during NASH progression. CK18 is detected by ELISA tests in patients suffering from NASH and it is significantly higher with respect to NAFLD patients [38,39]. Unfortunately, CK18 does not provide further information than the ALT transaminase, thus the gold standard diagnostic method to discriminate NAFLD from NASH is still the liver biopsy [2,37].

In the near future, innovative diagnostic methods currently under investigation might be employed to discriminate NASH from simple steatosis; for example, recent studies by Croce et al. have shown in animal models of NAFLD-NASH that liver tissue autofluorescence (AF) could be relevant for both experimental hepatology and clinical early diagnosis. By evaluating AF spectra of fatty acids, lipofuscin-like lipopigments (indices of oxidation), NAD(P)H and flavins (indices of energy and redox state), the author were able to discriminate steatosis from advanced NASH or fibrosis in methionine choline-deficient (MCD) and Zucker rat models [40]. The same authors also demonstrated that the spectrofluorometric analysis using a fiber-optic probe is an effective method for the in situ discrimination of fatty livers suitable for transplantation from those who would be too susceptible to cold ischemia/reperfusion injury, by assessing the differences in free fatty acids relative concentration [41]. In this work, livers underwent two different kinds of preservation techniques: cold storage and machine perfusion, followed by an ex-vivo model of liver reperfusion [42], suggesting that AF could be a suitable diagnostic method not only for applied research, but also in a clinical setting [41].

Different studies have pointed to asymmetric dimethylarginine (ADMA) as a potential serum marker for NAFLD diagnosis. ADMA is a physiological compound naturally produced during protein methylation; it acts as an unspecific, competitive inhibitor of nitric oxide synthases (NOS), and its levels are often seen to be increased in patients suffering from liver disorders [43], including NAFLD and NASH [44]. An excess of circulating ADMA inhibits nitric oxide (NO) production causing vasoconstriction, an increase in platelet aggregation, endothelium cell adhesion and vascular muscle cell proliferation [45]; so, changes in ADMA levels in NAFLD/NASH patients may induce further cardiovascular complications, the major cause of death in NAFLD patients, worsened by insulin resistance, hepatic dysfunction and chronic inflammation [46]. Moreover, changes in ADMA-NOS pathway play a pivotal role in the onset and progression of NAFLD and NASH in MCD diet-fed rats [47]. However, further clinical investigations are necessary to clarify whether ADMA is a good marker for the early diagnosis of NAFLD/NASH.

More recently, it has been found that large quantities of microparticles (MPs) are released by stressed/damaged hepatocytes, contributing to the occurrence of inflammation, fibrogenesis, and angiogenesis; consequently, MPs are considered another promising biomarker for NASH [48]. By evaluating MPs, it is possible to trace the cells that originated them, so they could be considered non-invasive biomarkers for NASH. In a murine model of NAFLD, blood MV levels were seen to be increasing in a time-dependent manner, becoming significantly higher after 8-week choline-deficient

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diet administration, a time point when early NAFLD symptoms become evident. The increase in MVs closely correlated with the histopathological findings. In addition, circulating MVs were enriched in miRNA-122 and miR-192-two microRNAs highly expressed in hepatocytes [49]. It has been shown that patients with NAFLD or NASH had high MV levels from natural killer (LK) lymphocytes and macrophages/monocytes; in those patients, MV correlated with severity of NASH (based on histology) [50]. Even though MPs derived from inflammatory cells are not specific for liver diseases, it has been shown that, using a proteomic approach, it is possible to tell apart MPs released because of free fatty acids oxidation-induced lipotoxicity, then originating from NASH [49].

Lastly, an interesting study has demonstrated that NASH could be diagnosed by the analysis of volatile organic compounds (VOCs) in the exhaled breath, using liquid chromatography and mass spectrometry. Verdam et al. studied VOCs from 65 obese patients undergoing bariatric surgery and liver biopsy and they found that three particular VOCs (n-tridecane, 3-methyl-butanonitrile, and 1-propanol) were typical of NASH and allowed to discriminate it from NAFLD. New analyses are needed to validate this method, but given its simplicity, it could be the election technique for NASH diagnosing in the near future [51].

5. Pathogenesis of NASH: Two-Hit versus Multiple-Hit Hypothesis

During the past years, different theories have been formulated about NAFLD onset and progression to NASH, from the traditional "two-hit" to the novel "multiple-hit" hypothesis. According to the two-hit hypothesis, the intrahepatic fat accumulation, triggered by sedentary lifestyle, bad nutritional habits and insulin resistance, represents the first hit [52]. The second hit consists in a lipid-induced over-production of reactive oxygen species; it worsens this scenario because of the interplay of various insults, such as: cytokine-mediated inflammation, free fatty acid oxidation, apoptosis, necroinflammation and fibrosis [53].

However, the two-hit hypothesis, by general consensus, is currently considered too simplistic to describe the complexity of human NASH development. In fact, it has been shown that oxidative stress does not necessarily follow lipid accumulation and it is able per se to induce steatosis [54,55]. Following this reasoning, it would be more correct to say that these events occur concurrently and they synergistically contribute to the development and progression of the disease as an integrated mechanism of different processes so-called "multiple-hit hypothesis" [2]. Hebbard & George explain the multiple-hit hypothesis as an "integrated response" of the organism to the combination of high-calorie diets, excessive food consumption and sedentary lifestyle in a genetically predisposed host. Altogether, these factors could lead to metabolic syndrome and obesity. After excessive food consumption, an imbalance of gut microbiota occurs and an increase in bacterial products is found in the portal circulation, activating the innate immune system. These events are accompanied by insulin resistance in the muscle, one of the key processes in the onset of the NAFLD-NASH, in response to the increased levels of circulating free fatty acids [56]. Insulin resistance causes the increase in hepatic de novo lipogenesis (DNL) and an imbalance in adipose tissue lipolysis, producing a great amount of circulating fatty acids that are conveyed to the liver. Moreover, insulin resistance causes the adipose tissue to release adipokines and inflammatory cytokines [57]. At the same time, hepatic fat accumulation leads to lipotoxicity, a condition promoting oxidative stress and affecting mitochondrial and endoplasmic reticulum physiological functions [58]. Altogether, these processes lead to hepatic chronic inflammation accompanied by cell death, hepatic stellate cell (HSC) activation and fibrosis (Figure 1). Thus, the assumption that steatosis always precedes inflammation is not completely correct, because the NASH can also be the initial hepatic injury: it is the timing and the combination of the various insults that determines whether steatosis or NASH will occur [59].

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